Paraffin sections (4 μm) were cut, dewaxed in xylene, and rehydrated in decreasing concentrations of ethanol (Apoteket Farmaci, Stockholm, Sweden). HCMV-IE and late proteins were detected in the tumor tissues by using an immunohistochemical (IHC) technique as previously described [23] (link), [28] (link). In negative control sections, the primary antibody was omitted. Cytokeratin and β-catenin served as staining controls. The following antibodies were used: anti–HCMV-IE and HCMV-LA antibodies (both from Chemicon, Temecula, CA), anti-cytokeratin antibody (DakoCytomation, Glostrup, Denmark), and anti–β-catenin antibody [BD Biosciences (Pharmingen), Stockholm, Sweden]. HCMV-infected lung tissue sections from a patient with human immunodeficiency virus (HIV) were used as a positive control for staining.
The number of HCMV protein–expressing cells in the tissue sections was estimated in one tissue section of the tumor specimen for each antibody (in serial sections). The sections were graded on the basis of the estimated percentages of IE- and LA-positive cells: negative (0% positive cells), low-grade infection (< 50% positive cells), or high-grade infection (≥ 50% positive cells). IHC staining was evaluated and graded by two investigators (A.R. and C.T.); neither had access to the clinical records of the patients. The results were confirmed by a pathologist (PR).
The number of HCMV protein–expressing cells in the tissue sections was estimated in one tissue section of the tumor specimen for each antibody (in serial sections). The sections were graded on the basis of the estimated percentages of IE- and LA-positive cells: negative (0% positive cells), low-grade infection (< 50% positive cells), or high-grade infection (≥ 50% positive cells). IHC staining was evaluated and graded by two investigators (A.R. and C.T.); neither had access to the clinical records of the patients. The results were confirmed by a pathologist (PR).